Biomedical subjects

J M Gleisner

Publications and source records attributed to J M Gleisner.

15 recordsLinked to original sources

PubMed1988

Effect of intravenous catalase on the pulmonary vascular response to endotoxemia in goats.

Neutrophils have been implicated in the pathogenesis of acute lung injury associated with clinical and experimental sepsis. Data from in vitro systems and experimental animals have suggested that neutrophil-derived oxidants, particularly H2O2, may be primarily responsible for endothelial damage, vasoconstriction, and lung edema. With the use of endotoxin infusion as an in vivo model of sepsis we tested the hypothesis that pretreatment with catalase, a peroxide scavenger, would ameliorate the resultant changes in pulmonary vasoconstriction and lung fluid balance. Paired experiments were performed in 16 goats with chronic lung lymph fistulas. One group of animals (n = 7) received endotoxin first alone and then again, several days later, after pretreatment with Ficoll-linked catalase. As a control, identical experiments were performed in a separate group (n = 6) with Ficoll-linked albumin substituted for Ficoll-catalase. A third group (n = 3) was given endotoxin alone and then again during a continuous infusion of catalase. Plasma and lymph levels of catalase were comparable to or exceeded those previously shown to be completely protective in isolated perfused lung preparations and in vitro systems. Endotoxin caused neutropenia, pulmonary arterial hypertension, decreased cardiac output, and increases in lymph flow to approximately three times base line, with a return of all variables toward control values by 6 h. Catalase pretreatment produced no significant differences in any of these variables. These experiments do not support a role for H2O2 as a mediator of acute lung injury due to endotoxemia.

Animals↗

PubMed1986

Lung tissue tension and glycosaminoglycans.

This study tested the hypothesis that the loss of tissue tension which occurs when lung parenchyma is immersed in phosphate buffered saline (PBS) follows a loss of glycosaminoglycans (GAG) and/or protein. Tissue tension decay (TTD) was examined in 30 X 30 X 100 micron strips of rat lung parenchyma immersed in PBS in the presence of agents altering the loss of GAG. GAG were labeled with [35S]sulfate in vitro by tissue culture of lung explants and in vivo by intraperitoneal injection of [35S]sulfate. Labeled GAG were lost from lung explants, lung homogenates, and whole lung immersed in saline. The rate of GAG loss was found to correlate with early TTD. In the presence of agents that markedly reduced the leach rate of GAG from lung parenchyma (Safranin O, Alcian Blue, Acridine Orange) there was no TTD. Cetyl-pyridinium chloride was also effective. Polybrene, however, caused a significantly greater TTD than seen in saline with some reduction in the total amount of GAG leached from the tissue. Greater amounts of heparin sulfate were lost in the presence of polybrene. Human serum, dextran-70, ovalbumin, and gelatin significantly reduced but did not prevent TTD. Dextran-70 was effective in reducing the loss of GAG from tissue explants. Agents that reduced disulfide bonds (dithiothreitol, 2-mercaptoethanol) markedly increased TTD and increased the loss of GAG and protein from lung explants and lung homogenates. These studies provide further evidence that lung tissue tension is a function of the interstitial matrix interacting with the connective tissue network. When the GAG and protein responsible for the swelling pressure of matrix are preserved the tissue tension is stable for one hour or more. Agents that partially preserve the matrix are somewhat effective in reducing TTD while those that increase the loss of GAG and protein increase the loss of tension. From the differential effect of agents that retard and accelerate GAG, heparin sulfate (HS) may be the most important GAG to the tissue properties.

Animals↗

PubMed1982

Structure of dihydrofolate reductase: primary sequence of the bovine liver enzyme.

The primary structure of dihydrofolate reductase from bovine liver has been established by Edman degradation of the intact carboxymethylated protein and of peptides obtained from the protein by the action of cyanogen bromide, trypsin, and the protease from Staphylococcus aureus, respectively. Since separation of some of the peptide mixtures by classical methods proved impossible, new systems were developed for the use of high-performance liquid chromatography to separate such mixtures. Some of the cleavage procedures used to obtain peptides gave atypical results at certain peptide bonds. The results are discussed in terms of the residues involved in these unexpectedly resistant or sensitive bonds. The sequence of the bovine liver enzyme is compared with those published for the enzyme from other sources, and known or probable functions of invariant residues are described. Sequences of vertebrate and bacterial reductases are compared and contrasted, and a possible role is considered for the residues which are invariant in bacterial reductases, but different in vertebrate reductases, in determining the selective inhibitory action of trimethoprim on bacterial reductases.

Amino Acid Sequence↗

PubMed1981

Inhibition of mast-cell degranulation by chemotactic peptides.

Pepstatin and f-methionyl peptides which are potent chemotactic agents for neutrophils were found to inhibit the increase in vascular permeability of rat skin which follows the injection of 48/80, anti-rat IgE serum, or pulmonary permeability factor. These latter compounds are known to act by releasing histamine from mast cells. f-Met-Leu-Phe, which is the most active chemotactically, was also found to be the most active inhibitor.

Animals↗

PubMed1981

Pulmonary polyamine permeability factor.

Acid extracts of calf lung have been found to contain low-molecular-weight factors which increase the permeability of the microcirculation when injected into the skin of rats. These factors, which were present in very low levels in aqueous extracts, were purified by gel filtration and ion-exchange chromatography. High-voltage paper electrophoresis revealed two active compounds with mobilities identical to the polyamines spermine and spermidine. Authentic samples of these compounds were as active in the "blueing" reaction as the isolated compounds. The permeability activity of both the isolated factors and the synthetic ones was inhibited by pepstatin and by pretreatment of the animals with pyrilamine maleate. If the normally low extracellular levels of these polyamines is increased by tissue damage, they could increase vascular permeability within the lung by releasing histamine from adjacent mast cells.

Animals↗

PubMed1981

Abnormal behavior of polyamines on gel filtration: a cautionary note.

The polyamines, spermine and spermidine, have been shown to persist in various tissue extracts despite procedures such as dialysis and ultrafiltration which normally remove such low molecular weight compounds. We have found that polyamines in tissue extracts and the standard compounds alone can migrate as much higher molecular weight compounds on gel filtration under a variety of conditions. Thus, even relatively pure fractions obtained from tissue extracts may be contaminated with, or consist entirely of, polyamines, which are potent inhibitors of cell proliferation under certain conditions.

Chromatography, Gel↗

PubMed1981

Low molecular weight inhibitors of lymphocyte transformation.

A variety of factors isolated from bovine thymus have been found to inhibit the transformation of human and mouse lymphocytes. The majority of this activity fractionates as low molecular weight material by ultrafiltration or column chromatography. Three distinct fractions of low molecular weight have been isolated. One fraction contains the polyamines spermine and spermidine. A second fraction contains thymidine or thymidine-like nucleotides. The third fraction appears to be polypeptide in nature, and gives an estimated molecular weight of 500--600, is heat and pH stable, and is easily extracted by solutions containing organic solvents. Preliminary steps in the isolation of this inhibitor are presented, and its relationship to other immunosuppressive and anti-mitotic agents is discussed.

Animals↗

PubMed1981

Characterization and comparison of aminopeptidase activity of various strains of Mycobacterium tuberculosis.

The aminopeptidase activity of three strains of Mycobacterium tuberculosis, H37Rv, H37Ra, and M. tuberculosis from a patient, was partially purified and characterized. The activity from all three organisms was found to be very similar, if not identical. All three aminopeptidases eluted at a similar salt concentration on DEAE Bio-Gel; were active on the same synthetic and peptide substrates; had molecular weights of 75-76,000; were found to be stable between pH 5 and 8, and 4 degrees and 40 degrees C; and had a pH optimum of 7. They were inhibited by low concentrations of Hg2+, Cu2+ and Co2+; metal chelators; and 4-chloromercuribenzoic acid. A number of amino acids and several antibiotics were also found to be inhibitory. Of the antibiotics tested, rifampicin and bacitracin were the most effective.

Aminopeptidases↗

PubMed1979

Macromolecular, anionic pulmonary permeability factor.

Aqueous extracts of fresh or acetone-powdered calf lung have been found to contain a factor which increases the permeability of the microcirculation when injected into the skin of rats. This permeability factor, which is not found in similar extracts of muscle or kidney, was concentrated by Amicon ultrafiltration at a molecular weight range of between 50,000 and 100,000 daltons and via isoelectric focusing at an IEP of pH 4.2. After preparative acrylamide gel electrophoresis, this permeability factor was homogeneous by electrophoretic criteria and in SDS acrylamide gel electrophoresis had a molecular weight of approximately 82,000 daltons. This apparently homogeneous permeability factor from lung was inhibited by pepstatin, and yet possessed no acid proteolytic activity against any substrate. Its activity was completely inhibited by pretreatment of the animals with antihistamines. This pepstatin-inhibitable permeability factor was found largely in the lysosomal fraction of fresh lung and could also be obtained by extraction of large amounts of alveolar "washout" macrophages. Since macrophages, during phagocytosis, are known to leak significant quantities of their lysosomal contents, the fact that one of these components is a material which could release histamine from mast cells and thereby increase locally the permeability of the microcirculation may be of importance in the defense system of the lung.

Animals↗

PubMed1975

Bovine liver dihydrofolate reductase: purification and properties of the enzyme.

A purification procedure is reported for obtaining bovine liver dihydrofolate reductase in high yield and amounts of 100-200 mg. A key step in the procedure is the use of an affinity gel prepared by coupling pteroyl-L-lysine to Sepharose. The purified reductase has a specific activity of about 100 units/mg and is homogeneous as judged by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and titration with methotrexate. The products of the first step of Edman degradation indicated a minimum purity of 79%. The reductase has a molecular weight of about 21500 on the basis of amino acid composition and 22100 +/- 300 from equilibrium sedimentation. It is not inhibited by antiserum to the Streptococcus faecium reductase (isoenzyme 2). Unlike the reductase of many other vertebrate tissues, the bovine enzyme is inhibited by mercurials rather than activated and it has a single pH optimum at both low and high ionic strength. However, the position of the pH optimum is shifted and the activity increased by increasing ionic strength. Automatic Edman degradation has been used to determine 34 of the amino-terminal 37 amino acid residues. Considerable homology exists between this region and the corresponding regions of the reductase from S. faecium and from Escherichia coli. This strengthens the idea that this region contributes to the structure of the binding site for dihydrofolate.

Amino Acid Sequence↗

PubMed1975

The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Amino acid sequence of peptide CNBr 7 and complete sequence of the protein.

The complete amino acid sequence of the mutant dihydrofolate reductase from Streptococcus faecium var. Durans strain A has been determined by sequence analysis of peptides produced by tryptic, chymotryptic, thermolytic, and mild acid cleavage of the large peptide CNBr 7 and from previously reported studies. The sequence of the S. faecium enzyme is compared to the reported sequence of dihydrofolate reductase from Escherichia coli and the two are shown to contain two domains of substantial homology. One of these domains consists of the NH2-terminal 60 residues and is considered to contribute the dihydrofolate binding site. The second domain probably contains the dinucleotide binding structure. Comparison of the sequences of the dihydrofolate reductases with those of larger dehydrogenases of known structure failed to show any evidence for homology. Considerations of size and predictions of secondary structure also suggest that the second domain in the reductases has no structural similarity to the nucleotide binding site in the larger dehydrogenases. It is concluded that the two reductases are related, although distantly, but that they have evolved from an ancestral protein different from the primitive predecessor of the other oxidoreductases.

Amino Acid Sequence↗

PubMed1975

The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Partial sequence and order of the limited tryptic and cyanogen bromide peptides.

The major form of dihydrofolate reductase from a methotrexate-resistant mutant (strain A) of Streptococcus faecium var. Durans has been purified on a large scale. Amino acid analysis of this form of the enzyme (isoenzyme 2) reveals an absence of cystine or cysteine, and sedimentation studies indicate a molecular weight of 20,800. The NH2-terminal sequence was determined by Edman degradation of the intact protein and the COOH terminus by selective tritiation and by carboxypeptidase treatment. After the action of trypsin on the citraconylated protein, seven of the expected nine peptides were purified from the digest, and after cyanogen bromide treatment of the unmodified protein, all seven of the anticipated peptides were isolated. The amino acid composition of all of these peptides has been established as well as their complete or partial sequences. From the results it was possible to order these peptides within the sequence and to establish the sequence of the NH2-terminal 60 residues and the COOH-terminal 11 residues.

Amino Acid Sequence↗

PubMed1975

The structure of dihydrofolate reductase. Identification of methionine residues carboxymethylated by iodoacetate with loss of catalytic activity.

Dihydrofolate reductase from the amethopterin-resistant mutant (strain A) of Streptococcus faecium var. Durans was reacted with iodo[14C]acetate according to three procedures; (a) in the absence of an inhibitor, (b) in the presence of aminopterin, and (c) in absence of inhibitor, but after treatment with unlabeled iodoacetate in presence of aminopterin. The first and last procedures resulted in the loss of approximately 90% of the catalytic activity, whereas in the presence of aminopterin essentially no activity was lost. Peptides were produced from all three labeled proteins by tryptic digestion after citraconylation of the lysine residues. From the amino acid compositions and partial amino acid sequences of these peptides the position of all modified methionines in the sequence was determined. The extent of labeling at each methionine, in enzyme labeled in the different procedures, indicated that methionines 28 and 50 may be at the binding site for inhibitors and that residue 50 is less accessible to iodoacetate than is residue 28. It is likely that carboxymethylation of residue 28 is responsible for the loss of enzyme activity.

Aminopterin↗

PubMed1975

The structure of dihydrofolate reductase. I. Inactivation of bacterial dihydrofolate reductase concomitant with modification of a methionine residue at the active site.

Carboxymethylation by iodoacetate of dihydrofolate reductase from the amethopterin-resistant mutant Streptococcus faecium var. Durans strain A leads to a loss of enzymic activity. Amino acid analysis showed that methionine is the only amino acid residue significantly affected by iodoacetate under the experimental conditions, and this was confirmed by the use of [1-14-C]iodoacetate and ion exchange chromatography of the products obtained by acid hydrolysis of the modified enzyme. During loss of 90% of the activity a total of about 2 of the 7 methionine residues present in the enzymes are carboxymethylated. Over this range of activity loss the decrease is proportional to the number of methionine residues modified. Fluorescence-quenching experiments demonstrated that dissociation constants for complexes of inhibitors with the carboxymethylated enzyme were 20 to 30 times greater than dissociation constants for corresponding complexes with native enzyme. Similarly, equilibrium dialysis studies showed that dihydrofolate binding to the modified enzyme was decreased 10-fold compared with binding to the native enzyme. These data suggest that iodoacetate modifies one or more methionine residues at the binding site for dihydrofolate and inhibitors. In accordance with this view it was shown that enzyme can be protected from inactivation by the folate analogue aminopterin and to a lesser extent by folate and dehydrofolate. Enzyme carboxymethylated in the presence of aminopterin, and subsequently freed of the latter, was found to bind inhibitors and dihydrofolate as tightly as the native enzyme. It is concluded that the loss of enzyme activity is caused by carboxymethylation of at least 1 methionine residue which is at or near the binding site of dihydrofolate.

Binding Sites↗

PubMed1974

Amino-acid sequence of dihydrofolate reductase from a methotrexate-resistant mutant of Streptococcus faecium and identification of methionine residues at the inhibitor binding site.

The amino-acid sequence of dihydrofolate reductase (7,8-dihydrofolate:NADP(+) oxidoreductase, EC 1.5.1.4) from S. faecium var Durans strain A is reported, and methionine residues 28 and 50 are shown to be protected by the inhibitor aminopterin from carboxymethylation by iodoacetate which occurs in absence of the inhibitor. Comparison of the sequence with that of the Escherichia coli reductase reveals two domains of considerable homology, one (the N-terminal region) presumably concerned with dihydrofolate and inhibitor binding and the other with dinucleotide binding. No significant sequence homology was found between larger dehydrogenases and the dihydrofolate reductases, which must, therefore, have evolved from a different ancestral protein.

Amino Acid Sequence↗